1. Field of the Invention
This invention relates to a method of determining drug levels of body fluids by micellar chromatography. More specifically, this invention relates to the determination of the concentrations of drugs in bodily fluids, i.e. serum or urine by direct injection into a micellar chromatographic column.
2. Discussion of the Prior Art
Therapeutic drug monitoring has shown rapid growth recently due to the development of specific and sensitive assays for minute quantities of drugs in various body fluids. In addition, therapeutic drug monitoring has become an essential part of many hospital clinician's work, i.e. physicians, medical technicians and nurses.
Normally, therapeutic drug monitoring is considered when several of the following conditions exist: (1) there is a question of patient compliance, (2) there is a lack of therapeutic effect, (3) the drug has a narrow therapeutic range, (4) there is a danger of toxicity and (5) there is a need for medico-legal verification of treatment.
Various techniques are used in therapeutic drug monitoring, such as, radioimmunoassay, flourescence immunoassays and enzyme-multiplied immunoassay techniques. Additionally, chromatographic methods such as thin layer chromatography, gas-liquid chromatography and high performance liquid chromatography are widely used.
The use of radioimmunoassay techniques for therapeutic drug and toxic drug level monitoring remains the most popular technique. Foremost among these are the Abuscreen kits, which are illustrated by the barbiturate test kit. The Abuscreen radioimmunoassay kit for barbiturates is based on the competitive binding to an antibody of radiolabeled antigen and unlabeled antigen in proportion to their concentration in the solution. Unlabeled antigen displaces radioactive antigen from the antibody present. An unknown specimen is added to a test tube containing known amounts of barbiturate antibodies and radiolabeled antigen. After precipitation and centrifugation, the supernatant fluid, which contains the free antigen, is transferred to test tubes for counting in a gamma scintillation counter. A positive specimen is identified qualitatively when the radioactivity is equal to or greater than that of the positive control and quantitatively by comparison to a standard curve. These radioimmunoassay techniques have been applied to various drugs including morphine, cocaine, amphetamine, methadone hydrochloride, and phencylidine phenol among others.
As noted, up to the present time, the radioimmunoassay (RIA) method in its various forms has been the most sensitive system available. The RIA method, unfortunately, has several serious disadvantages, including the requirement of special equipment, trained staff, the recited need for extra safety measures to protect against harmful radiation, special licensing, controlled radioactive waste disposal and the continuous disappearance of labeled compound by radioactive decay. The possibility of replacing the radioactive label with an enzyme label was proposed in 1968 in an article by L. E. M. Miles and C. N. Hales, entitled "Labelled Antibodies and Immunological Assay Systems", Lancet, II, 492 (1968), and Nature 219, 168 (1968). No procedural details were provided; the article offered only the general idea, leaving it to future workers to determine the basic step and to perform the extensive experimentation needed to establish a practical operative enzymatic immunoassay method.
Various chromatographic techniques have been utilized for different types of therapeutic drug monitoring. For example, thin layer chromatography is used to screen urine specimens for drugs of abuse. Many prepackaged kits are available for this purpose; the kits contain plates, sprays, applicators, and reference tables for determining color reactions and R.sub.f (distance ratio) values for the compounds of interest. The most common drugs screened with this type of procedure are salicylates, barbiturates, opiates, pentachlorophenol, benzodiazepines and amphetamines. It is recommended that positive results obtained should be verified with other methodologies. A urine sample is extracted with an organic solvent and a concentrated aliquot is applied in a small spot to a thin layer chromatography plate which is coated with cellulose or another coating material. The plate with the dried spots is immersed in a tank containing a mobile phase, the mobile phase moves up the thin layer chromatography plate by capillary action, and the constituents of the mixture contained in the spot are carried across the plate with the mobile phase. In transit, they partition between the solvent and the particles of the coating. The partition retards some constituents and allows others to move more rapidly in comparison with the solvent front. The plate is dried after the solvent has moved a sufficient distance up the plate. A series of sprays are applied and characteristic colors of the mobilities are observed for compounds of interest.
A publication by Paul Yarmchuk, et al., entitled "Selectivity in Liquid Chromatography with Micellar Mobile Phases" in Anal. Chem. 1982, Vol. 54, 2233-2238 describes the use of micellar mobile phases to control selectivity in liquid chromatography. The study compared two surfactants which form comparable micelles differing only in the nature of the polar head groups. The surfactants studied were sodium lauryl sulfate and dodecyltrimethylammonium bromide. Results of the study illustrated that selectivity can be enhanced by proper choice of surfactant type and mobile phase concentration.
Another publication by Daniel W. Armstrong, et al., entitled "Evaluation and Pertubation of Micelle-Solute Interactions", J. Am. Chem. Soc., 1983, Vol. 105, pp. 6220-6223 describes the interaction of seven compounds with sodium dodecyl sulfate micelles using LC and TLC. Results of the study illustrated that the solute-micelle interaction can be classified as binding, nonbinding, or antibinding.
A publication by Daniel W. Armstrong, et al., entitled "Selectivity in Pseudophase Liquid Chromatography", Anal. Chem., Vol. 55, pp. 2317-2320 describes the pseudophase liquid chromatographic separation for fourteen compounds by using anionic micellar mobile phase. The study discussed the different characteristics of the compounds using the pseudophase liquid chromatography.
While the art has provided various methods for therapeutic drug monitoring, the need still exists for a method of therapeutic drug monitoring that is accurate and utilizes more sensitive chromatographic techniques. For example, present chromatographic methods generally require separation of the drug from the serum's protein base before analysis. This preparation step involves tedious and time consuming extraction procedures and/or protein precipitation steps. Accordingly, it is one object of the present invention to provide a novel method of therapeutic drug monitoring utilizing chromatographic methods.
Another object of the present invention herein is to provide a new method of therapeutic drug monitoring utilizing micellar chromatography.
A further object of this invention is to provide a new method of therapeutic drug monitoring that allows direct injection of a bodily fluid into a chromatographic column.
Still another object of this invention is to provide a new method of determining the concentration and identifying analytes in a bodily fluid utilizing micellar chromatography.
A still further object of this invention is to provide an improved method of therapeutic drug monitoring that involves direct injection of body fluids without any preparation or extraction of the sample.
The achievement of these and other objects will be apparent from the following description of the subject invention.